Glycosyltransferases constitute a superfamily of enzymes that are involved in the synthesis of complex carbohydrates present on glycoproteins and glycolipids. The fundamental role of a glycosyltransferase is to transfer the glycosyl moiety of a nucleotide derivative to a specific sugar acceptor. β-1,4-Galactosyltransferases (β4GalTs) (EC 2.4.1.38) constitute one of the subfamilies of glycosyltransferase superfamily—comprising at least seven members Gal-T1 to Gal-T7—which catalyze the transfer of galactose (Gal) from UDP-Gal to different sugar acceptors. A common motif resulting from a galactose transferase onto a terminal GlcNAc residue is the lactosamine sequence Galβ4GlcNAc-R (LacNAc or LN), which is subsequently modified in a variety of ways by the additions of other sugars and sulfate groups. The most common and important sugar structure of membrane glycoconjugates is poly-N-acetyllactosamine (poly-LN), which linked to proteins (or lipids), plays an important role in cellular communication, adhesion, and signalling and are key molecules in regulation of immune responses.
Another common terminal motif found in vertebrate and invertebrate glycoconjugates is the GalNAcβ4GlcNAc-R (LacdiNAc or LDN) sequence. The LDN motif occurs in mammalian pituitary glycoprotein hormones, where the terminal GalNAc residues are 4-O-sulfated and function as recognition markers for clearance by the endothelial cell Man/S4GGnM receptor. However, non-pituitary mammalian glycoproteins also contain LDN determinants. In addition, LDN and modifications of LDN sequences are common antigenic determinants in many parasitic nematodes and trematodes. The biosynthesis of LDN involves the transfer of GalNAc to a terminal GlcNAc, a process executed by highly specific GalNAc-transferases. For example it was reported by Miller et al. in J. Biol. Chem. 2008, 283, p. 1985, incorporated by reference, that two closely related β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, are thought to account for the protein-specific addition of β1,4-linked GalNAc to Asn-linked oligosaccharides on a number of glycoproteins including the glycoprotein luteinizing hormone (LH) and carbonic anhydrase-6 (CA6).
β-(1,4)-Acetylgalactosaminyltransferases (β-(1,4)-GalNAcTs) have been identified in a range of organisms, including humans, Caenorhabditis elegans (Kawar et al., J. Biol. Chem. 2002, 277, 34924, incorporated by reference), Drosophila melanogaster (Hoskins et al. Science 2007, 316, 1625, incorporated by reference) and Trichoplusia ni (Vadaie et al., J. Biol. Chem. 2004, 279, 33501, incorporated by reference).
Finally, besides GalTs and GalNAcTs involved in N-glycoprotein modification, a non-related class of enzymes called UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (also referred to as ppGalNAcTs) is responsible for the biosynthesis of mucin-type linkages (GalNAc-α-1-O-Ser/Thr). These enzymes transfer GalNAc from the sugar donor UDP-GalNAc to serine and threonine residues, forming an alpha anomeric linkage typical in O-glycoproteins. Despite the seeming simplicity of ppGalNAcTs catalytic function, it is estimated on the basis of in silico analysis that there are 24 unique ppGalNAcTs human genes alone. Because O-linked glycosylation proceeds step-wise, addition of GalNAc to serine or threonine represents the first committed step in mucin biosynthesis. Despite this seeming simplicity, multiple ppGalNAcTs family members appear to be necessary to fully glycosylate their protein substrates.
It has been shown that galactosyltransferases are able to transfer, besides the natural substrate UDP-Gal, a range of unnatural galactose derivatives to an acceptor GlcNAc substrate. For example, Elling et al. in ChemBioChem 2001, 2, 884, incorporated by reference, showed that terminal GlcNAc-containing proteins can be biotinylated by transfer of a 6-modified galactose from an UDP-sugar under the action of a range of galactosyltransferases. Similarly, it was demonstrated by Pannecoucke et al. in Tetrahedron Lett. 2008, 49, 2294, incorporated by reference, that 6-azido-6-deoxygalactose can be transferred (to some extent) from the corresponding UDP-sugar to a small molecule GlcNAc substrate upon subjection to bovine β1,4-galactosyltransferase. The use of glycosyltransferases for modified galactose derivatives was also reported earlier in US 2008/0108557 (WO 2006/035057, Novo Nordisk A/S), where it is claimed that a wide range of galactose derivatives modified at C-6 (e.g. thiol, azide, O-propargyl, aldehyde) can be transferred to a GlcNAc substrate under the action of (bovine or human) β1,4-galactosyltransferase, using 2-10 equivalents of UDP-sugars. However, the data provided to support such claims concern only the 6-O-propargyl and 6-aldehydo variant of galactose. A range of GalNAc derivatives with a chemical handle at C2 is also claimed as substrates for glycosyltransferases but no examples were provided.
In particular the mutation of the Tyr-289 residue to Leu-289 in bovine β4Gal-T1, as reported by Ramakrishnan et al. J. Biol. Chem. 2002, 23, 20833, incorporated by reference, creates a catalytic pocket of the enzyme that can facilitate a UDP-Gal molecule carrying a chemical handle at C2, such as 2-keto-Gal. By a two-step procedure involving first transfer of the unnatural galactose moiety followed by oxime ligation onto the C-2 handle, this mutant enzyme, β4GalT(Y289L), has been used for in vitro detection of O-GlcNAc residues on proteins or the presence of a terminal GlcNAc moiety on the cell surface glycans of normal and malignant tumor tissues.
For example Khidekel et al., J. Am. Chem. Soc. 2003, 125, 16162, incorporated by reference, discloses chemoselective installation of an unnatural ketone functionality to O-GlcNAc modified proteins with β4GalT(Y289L). The ketone moiety serves as a unique marker to “tag” O-GlcNAc glycosylated proteins with biotin using oxime ligation. Once biotinylated, the glycoconjugates can be readily detected by chemiluminescence using streptavidin conjugated to horseradish peroxidase (HRP).
For example WO 2007/095506, WO 2008/029281 (both Invitrogen Corporation), WO 2014/065661 (SynAffix B.V.) and Clark et al. J. Am. Chem. Soc. 2008, 130, 11576, all incorporated by reference, report a similar approach, using β4GalT(Y289L) and azidoacetyl variants of galactosamine, with similar success.
For example U.S. Pat. No. 8,697,061 (Glykos), incorporated by reference, reports a similar approach, using β4GalT(Y289L) and 2-modified sugars, with similar success.
Mutant β4GalT(Y289L) has also been applied most recently in a preparative fashion for the site-selective radiolabeling of antibodies on the heavy chain glycans, as reported by Zeglis et al. in Bioconj. Chem. 2013, 24, 1057, incorporated by reference. In particular, the incorporation of azide-modified N-acetylgalactosamine monosaccharides (GalNAz) into the glycans of the antibody allowed the controlled labeling with 89Zr upon after click chemistry introduction of the appropriate chelator.
Ramakrishnan et al. in Biochemistry 2004, 43, 12513, incorporated by reference, describe that the double mutant β4GalT(Y289L,M344H) loses 98% of its Mn2+-dependent activity, but nevertheless shows 25-30% activity in the presence of Mg2+, including a capability to transfer C-2 modified galactose substrates. The double mutant β4GalT(Y289L,M344H) was found useful for in vitro galactosylation assays, because the typical requirement of 5-10 mM Mn2+ is known to have potential cytotoxic effects for the cells.
Mercer et al., Bioconjugate Chem. 2013, 24, 144, incorporated by reference, describe that a double mutant Y289L-M344H-β4Gal-T1 enzyme transfers GalNAc and analogue sugars to the acceptor GlcNAc in the presence of Mg2+.
Attempts to employ a wild-type β-(1,4)-N-acetylgalactosaminyltransferase, herein also referred to as β-(1,4)-GalNAcT, for the transfer of C-2 modified GalNAc have met little success to date.
Bertozzi et al. in ACS Chem. Biol. 2009, 4, 1068, incorporated by reference herein, applied the bioorthogonal chemical reporter technique for the molecular imaging of mucin-type O-glycans in live C. elegans. Worms were treated with the azido-sugar variant of N-acetylgalactosamine (GalNAz) allowing the in vivo incorporation of this unnatural sugar. Although metabolic incorporation of GalNAz into glycoproteins was observed, both chondroitinase ABC and peptide N-glycosidase F (PNGase F) digestion of C. elegans lysate, followed by the Staudinger ligation using a phosphine-Flag tag and subsequent probing of the glycoproteins by Western blotting utilizing an α-Flag antibody, indicated that the majority of GalNAz residues on glycoproteins were situated in other types of glycans than N-glycans. In addition, no detectable binding of azide-labeled glycoproteins to the N-glycan specific lectin concanavalin A (ConA) was observed, consistent with the hypothesis that the vast majority of labelled glycans are O-linked and not N-linked. Based on these observations, one may conclude that GalNAz does not metabolically incorporate onto N-GlcNAcylated proteins in this organism.
A similar conclusion was drawn most recently by Burnham-Marusich et al. in Plos One 2012, 7, e49020, incorporated by reference herein, where lack of signal reduction upon PNGase treatment—indicating no apparent incorporation of GalNAz in N-glycoproteins—was also observed. Burnham-Marusich et al. describe a study using the Cu(I)-catalyzed azide-alkyne cycloaddition reaction of a terminal alkyne-probe with an azido-labeled glycoprotein to detect metabolically labelled glycoproteins. Results indicated that the majority of the GalNAz label is incorporated into glycan classes that are insensitive to PNGase F, hence are not N-glycoproteins.
High substrate specificity of a β-(1,4)-GalNAcT for UDP-GalNAc becomes apparent from the poor recognition of UDP-GlcNAc, UDP-Glc and UDP-Gal, for which only 0.7%, 0.2% and 1% transferase activity remains, respectively, as was reported by Kawar et al., J. Biol. Chem. 2002, 277, 34924, incorporated by reference.
Based on the above, it is not surprising that no in vitro method for modification of glycoproteins has been reported by means of GalNAc-transferase of an unnatural GalNAc derivative such as a 2-keto or 2-azidoacetyl derivative.
At the same time, it has been reported by Qasba et al., J. Mol. Biol. 2007, 365, 570, incorporated by reference, that substitution of the Ile or Leu active site residue in invertebrate GalNAcTs—corresponding to the Tyr-289 residue in human β4Gal-T1 ortholog enzymes—for a Tyr residue, converts the enzyme to a β(1,4)galactosyltransferase by reducing its N-Acetylgalactosaminyltransferase activity by nearly 1000-fold, while enhancing its galactosyltransferase activity by 80-fold.
Taron et al., Carbohydr. Res. 2012, 362, 62, incorporated by reference, describe the in vivo metabolic incorporation of GalNAz in GPI-anchors.